JPH04218368A - Production of plasminogen activator precursor - Google Patents

Abstract

PURPOSE:To obtain the subject precursor in high purity by culturing a cell capable of producing a plasminogen activator precursor, partially purifying the supernatant of the culture product by ion-exchange chromatography, eluting the adsorbed precursor with a buffer solution and contacting the eluted liquid with a precursor antibody column. CONSTITUTION:The objective plasminogen activator precursor is produced by successively performing at least the following steps (a) to (d). (a) Supernatant liquid of culture product of a cell capable of producing the subject precursor is made to contact with a cation exchange material at pH4.5-6.5 to effect the adsorption of the precursor. (b) The adsorbed precursor is eluted from the cation exchange material at pH7.5-9.5. (c) The eluted fraction is made to contact with the above precursor antibody column at pH6-8 to effect the adsorption of the precursor. (d) The adsorbed precursor is eluted from the antibody column at pH2-4.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing a plasminogen activator precursor.

[Prior Art/Problem to be Solved by the Invention] Plasminogen activator is an activator that converts plasminogen into active plasmin having fibrinolytic ability.

Hitherto, urokinase has been well-known as a plasminogen activator. This product is purified from human urine and human kidney cell culture fluid, and mainly consists of two types, one with a molecular weight of 30,000 and the other with a molecular weight of 50,000. High molecular weight urokinase has high activity and is most useful as a medicine.

Its molecular structure consists of two chains, the H chain (molecular weight 30,000) and the L chain (molecular weight 20,000), which are connected only by a disulfide bond. Therefore, it had the property of being easily reduced to a lower molecular weight by reduction treatment.

The present inventor recognized the above-mentioned findings and conducted research to obtain a plasminogen activator precursor (hereinafter also referred to as "zymogen") having better plasminogen activation ability. As a result, the inventors discovered a method for separating and purifying zymogen, which has a higher affinity for fibrin than the known molecular type of urokinase, and completed the present invention.

[Means to solve the problem]

That is, the present invention is a method for producing a plasminogen activator precursor, which comprises at least the following (a) to (d).
) is characterized by sequentially performing the steps.

(a) Cell culture supernatant producing this precursor at pH 4.5~
The present precursor is adsorbed by contacting with a cation exchanger under the conditions of 6.5.

(b) The present precursor is eluted from the cation exchanger under conditions of pH 7.5 to 9.5.

(c) The eluted fraction is brought into contact with the present precursor antibody column under conditions of pH 6 to 8 to adsorb the present precursor.

(d) The precursor is eluted from the antibody column under conditions of pH 2 to 4.

Furthermore, especially when the cells producing this precursor are human kidney cells, a zymogen having the following properties can be produced by the method of the present invention.

(a) The molecular weight measured by SDS-polyacrylamide gel electrophoresis is approximately 50,000 Daltons.

(a) Low molecular weight does not occur due to reducing agent treatment.

(c) Zymogen itself does not exhibit enzymatic activity.

(d) Enzyme activity is expressed by plasmin treatment, and the specific activity at that time is at least 80,000 IU/mg.

When carrying out the method of the present invention, cells that produce plasminogen activator precursors are prepared and cultured as follows.

<Cell Preparation> The cells are not particularly limited as long as they produce plasminogen activator precursors, but human kidney cells are preferably used. The human kidney cells used include, for example, primary culture or dipoid cells obtained from human fetal kidney, which are subcultured and the zymogen-producing cells are isolated.

For example, cells are implanted at a number of 2 to 20 x 104 cells/ml, cultured for about 3 days, and when the number of cells becomes about three times the number of implanted cells, a trypsin-EDTA mixture is added to form a monolayer of young cells. What is obtained by collecting cells is used.

<Culture conditions> Examples of the culture medium include Waymouth's medium,
Dulbecco's modified MEM medium or the like is used, and 5% heat-inactivated tallow serum is added to the medium during pre-culture, and a serum-free medium, preferably a serum-free medium supplemented with human serum albumin, is used during production of the zymogen. Culture using serum medium. Human or bovine albumin, lactalbumin hydrolyzate, transferrin, various amino acids, various fatty acids, hormones such as insulin, etc. may be added to the serum-free medium. The culture medium is replaced every 2 to 3 days.

The subject zymogen can be produced in this medium.

The produced zymogen is recovered (separated and purified) as follows.

<Recovery of the present zymogen> The present zymogen can be recovered from the culture medium by, for example, centrifuging the culture medium, vacuum concentration, salting out fractionation, gel filtration, concentration, ion exchange chromatography, affinity chromatography, etc., in an appropriate combination. It is carried out by

More specifically, it is recovered by the following method, for example. That is, first, the medium is centrifuged and the supernatant is collected. This recovered solution is partially purified by ion exchange chromatography. As a carrier, a weakly acidic cation exchanger is most suitable, such as CM-exchanger or Duoli
te etc. are exemplified. After adjusting the carrier to pH 4.5 to 6.5, more preferably pH 5 to 6, the recovered liquid is developed and adsorbed onto the carrier.

After washing with the above buffer, the zymogen is eluted with a buffer having a pH of 7.5 to 9.5, more preferably a pH of 8 to 9. Examples of the buffer include phosphate buffer and the like. Furthermore, this eluate is highly purified by affinity chromatography. As the carrier, either a polyclonal antibody column or a monoclonal antibody column may be used.

In the case of the polyclonal method, the anti-zymogen antibody is obtained by collecting and purifying the serum obtained by immunizing an animal with the highly purified zymogen.

The antiserum may be produced by a known method; for example, a mixed emulsion of highly purified zymogen and Freund's complete adjuvant is prepared, injected intradermally into the animal 2 to 3 times, and blood collected several days after the final immunization. After solidifying at room temperature, 4
The antiserum is obtained by standing overnight at ℃ and centrifuging at 3,000 rpm for 20 minutes.

It is not necessary to select a particular animal species as the animal used for immunization, and examples thereof include rats, mice, rabbits, goats, horses, and the like. The purification of the antiserum is described, for example, in J.
Am. Chem. Soc. , 62.3386 (1940
), Fed. Proc. , 17.1161 (1958)
This is done using the method described in .

In the case of the monoclonal method, the anti-zymogen is obtained by cell fusion method. The cell fusion method is carried out by means known per se,
One example is to react proliferative cells with lymphocytes that produce the desired antibody in the presence of polyethylene glycol to produce cells that have proliferative and antibody-producing abilities at the same time. The antibody produced by these cells is a single antibody that reacts with only one antigenic determinant.

In the present invention, mouse myeloma cells are used as proliferative cells and mouse spleen cells (B cells) immunized with the present zymogen are fused as antibody-producing lymphocytes, and cells producing the target antibody are fused. A monoclonal antibody against the zymogen is obtained by screening.

Furthermore, as a method for immobilizing the anti-zymogen antibody thus obtained without losing its activity, the following insoluble matrix can be applied. Copolymers of amino acids (J. Biol. Chem., 236, 1
970 (1961)), cellulose (Nature, 1
89, 576 (1961)), agarose or Sephadex (Nature, 215, 1491 (196
7), Nature, 245, 3059 (1970))
, polyacrylamide (Bio-chem., 8.40
74 (1966)). By these methods, the anti-zymogen antibody can be efficiently immobilized. Furthermore, by using the adsorbent thus obtained, the present zymogen can be obtained in good yield and with high purity.

Affinity chromatography of this zymogen is as follows. The present zymogen partially purified using a cation exchanger is brought into contact with an anti-present zymogen antibody column equilibrated with a buffer solution of pH 6 to 8, and adsorbed thereon. After adsorption, the column is washed and elution is performed with an aqueous solution of pH 2-4.

Note that the above-mentioned collection method is merely an example of the method of the present invention, and of course, collection may be performed by adding other methods. For example, since the antigenicities are identical, a carrier on which an anti-urokinase antibody is immobilized can be similarly used for purifying the present zymogen. The thus obtained zymogen can be used for chemical purposes,
It may be used as a pharmaceutical or medical reagent, and when used as a pharmaceutical, heat treatment, sterile filtration, freeze-drying, dispensing, and
All you have to do is formulate it into a formulation. Also, during or after the purification process,
Albumin or nonionic surfactants, such as Triton X-100, Tween 8, as stabilizers in the solution.
It is preferable to add 0 or the like.

Thus, useful medicaments containing the present plasminogen activator precursors are provided.

The properties of an example of the blasminogen activator precursor (examples described below) obtained by the method of the present invention are shown below.

<Characteristics of this zymogen> ■Molecular weight SDS-polyacrylamide gel electrophoresis method (Natu
re. 227.680-685 (1970)), the molecular weight of the zymogen obtained by the method of the present invention was determined to be approximately 50,000 Daltons. The molecular weight was determined by comparison with a standard protein of known molecular weight, and pretreatment was performed at 37°C for 2 hours or at 100°C for 2 minutes at 1%
Each sample was subjected to reduction treatment with 2-mercaptoethanol.

■Enzyme sensitivity J. Biol. Chem. , 257.3276-328
3 (1980), sensitivity experiments to plasmin were conducted. As a result, the present zymogen itself did not exhibit plasminogen activator activity. However, activity was expressed by plasmin treatment, and the degree of activity expression depended on the concentration of plasmin treatment (Table 1) and the treatment time (Table 2). The activity measurement method is as described below.

In the former experiment, the amount of this zymogen protein was 1.3 μg/
m■ is prepared and added to it by approximately 6 m
The enzyme activity expressed after 0 minutes of pretreatment was measured.

In the latter experiment, plasmin was prepared at 0.1 μg/m2 and the amount of the present zymogen protein was 1.3 μg/m2, and the effect of treatment with plasmin was measured over time.

This revealed that the present plasminogen activator precursor is a type of zymogen.

Furthermore, when this zymogen was treated with plasmin and then subjected to reduction treatment and subjected to SDS-polyacrylamide gel electrophoresis, it was found that it was decomposed into fragments with a molecular weight of about 30,000 and a molecular weight of about 20,000. Therefore, the product after plasmin treatment is presumed to be the same as conventional human urine-derived urokinase.

(Left below) ■Reducing agent treatment 1% SDS, 1% 2-mercaptoethanol, 37°C.
The resistance of this zymogen to treatment for 2 hours or 2 minutes at 100° C. was investigated according to a standard molecular weight method. As a result, the untreated zymogen and the treated zymogen showed the same single band electrophoresis pattern, confirming that the zymogen was a single chain.

■Activity measurement method Synthetic substrate method (Cleason et al. Haemostasis.
776 (1978)) or the plate method (Astrup et al. Arch.Biochem.Biophys..40
, 346-351 (1952)). Fibrinogen is bovine from Miles.
．． fibrinogen. Fr. I (containing trace amounts of plasmin) was used.

■Amino acid composition and sequence The present inventors have already discovered m
RNA was isolated from the same human kidney cells used in the present invention, and the base sequence of the cDNA was determined (Japanese Patent Application No. 1983-3).
No. 7119). On the other hand, this zymogen was chemically cleaved using CNBr, lysyl endopepti-dase
For each fragment obtained by enzymatic cleavage using Abride Biosystems' Gas-Pha
se Protein Sequencer Mode
By automated Edman decomposition method using l 470A,
The amino acid sequence of a region of 350 amino acid residues (85% of the total structure) was determined and is shown in FIG. As a result, the amino acid sequence predicted from the cDNA encoding the human urokinase precursor and the amino acid sequence of this zymogen were completely identical.

Furthermore, the present zymogen was hydrolyzed and its amino acid composition was investigated (Table 3). The amino acid composition of both samples was also consistent.

These findings strongly support that the present zymogen corresponds to the urokinase precursor itself deduced from the cDNA encoding human urokinase precursor (human UK precursor).

■Other properties Active center: p binds to the serine active site of urokinase
-This zymogen was brought into contact with Sepharose gel on which aminobenzamidine was immobilized, but it was not adsorbed. From this, it is presumed that the serine active site of this zymogen is located inside the molecule, and its higher-order structure is different from that of conventional urokinase.

Secondary structure: When the α-helix content of this zymogen was examined by circular dichroism, it was found that the α-helix content was higher than that of conventional human urine-derived urokinase. This indicates that the present zymogen and conventional human urine-derived urokinase have different secondary structures.

Fibrin affinity: The zymogen (5 U as enzyme amount) was added to a reaction mixture (eg plasma) containing 2 mg/ml of fibrinogen. This specimen was coagulated with thrombin and then incubated at 37°C for 15 minutes. The clot and supernatant were centrifuged, and the plasminogen activity of the supernatant was measured. This value was taken as the unbound amount, and the value subtracted from the total amount was calculated as the bound amount of fibrin (Table 4).

This zymogen has a strong affinity for fibrin and has properties similar to tissue plasminogen activator. This has important implications in thrombolytic therapy. That is,
With conventional urokinase, plasmin deactivates quickly, so large doses must be administered, which causes serious side effects such as bleeding tendency. However, since this zymogen has a high affinity for fibrin, it can induce a fibrinolytic phenomenon limited to the solid phase (fibrin), providing an ideal drug for thrombolytic therapy.

Effect on fibrinogen: Conventional human urine-derived urokinase is used to treat fibrinogen in plasma, coagulation factors (factor V, factor VIII,
It also degrades factor III), and the side effect of increased bleeding tendency becomes a problem. Therefore, we investigated the decomposition of fibrinogen in plasma by this zymogen.

Fibrinogen labeled with 125I was added in advance to human plasma, and the present zymogen (500 U/ml) or conventional human urine-derived urokinase (1,500 IU/ml) was added to human plasma.
) was added, and samples were taken after 2 minutes, 10 minutes, 60 minutes, 120 minutes, and 180 minutes at 37°C, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.
The degree of degradation of 125I-fibrinogen was measured over time.

Fibrinogen with a molecular weight of 330,000 has a molecular weight of 240,000, 155,000, 8
Although it is broken down into 5,000, 50,000 fragments, and other smaller fragments, this zymogen has 180 fragments in plasma.
Even when present for minutes, fibrinogen was hardly degraded. On the other hand, when conventional human urine-derived urokinase was applied, a considerable amount of fibrinogen was degraded within 10 minutes, and the degradation progressed further.

In other words, although this zymogen has a high affinity for fibrin and a high fibrinolytic ability, it does not degrade fibrinogen in plasma, so it only degrades fibrin at the site of thrombus, causing side effects that can be a problem when administering large amounts of urokinase. It can be said that it is unlikely to cause an increase in bleeding tendency due to a decrease in blood fibrinogen.

Thrombolytic ability: The ability to dissolve fibrin clots prepared from human plasma was investigated. The fibrin thrombus labeled with 125I was left at 37°C for 3 hours in plasma containing the present zymogen or conventional human urine-derived urokinase, and the radioactivity of the dissolved fibrin was measured. The results are shown in Table 5.

The results revealed that the thrombolytic ability of the present zymogen was approximately three times superior to that of conventional human urine-derived urokinase.

Stability in plasma: The stability of this zymogen in plasma was investigated by examining its molecular weight and single-chain structure. This zymogen labeled with 125I (500U/ml)
was left in human plasma at 37°C, sampled after 1 hour, 2 hours, and 3 hours, and divided into two halves. One is 1%S
One was denatured with DS, and the other was subjected to reduction treatment with 1% SDS and 1% 2-mercaptoethanol. As a result of these SDS-polyacrylamide gel electrophoresis and autoradiography, even after 3 hours, both non-reduced and reduced treatments showed the same migration pattern as at 0 hours, showing a single band with a molecular weight of about 50,000. Ta. Therefore, the molecular weight and single-chain structure of this zymogen can be said to be stable in plasma.

The urokinase activity of this zymogen in plasma was measured using the synthetic substrate method described above, but no activity was detected.

From the above, it can be said that the present zymogen is stable in plasma as an enzyme precursor.

Anti-urokinase antibody and anti-human melanoma-derived TPA
(Tissue plasminogen activator) Neutralization of enzyme activity using antibody: The activity of this zymogen was expressed using plasmin. Furthermore, anti-urokinase antibody or anti-human melanoma-derived TPA antibody was added, and
After standing for a minute, the remaining enzyme activity was measured by the synthetic substrate method or the plate method, and the enzyme activity of this zymogen expressed by plasmin treatment was inhibited by the anti-urokinase antibody, but not by the anti-TPA antibody. Ta.

From the above, the present zymogen is a precursor of urokinase, and although it exhibits properties similar to TPA in terms of fibrin affinity, it is a substance different from TPA and its precursor.

Mechanism of thrombolysis: From the above properties of the present zymogen, it is thought that this enzyme has a thrombolytic mechanism different from that of conventional human urine-derived urokinase.

Urokinase derived from human urine directly acts on plasminogen in plasma and on blood clots to produce plasmin, which degrades fibrinogen and fibrin.

On the other hand, this zymogen does not show plasminogen activator activity in plasma and has a high affinity for fibrin, so it easily reaches the site of thrombus, binds to fibrin, and is activated by a trace amount of plasmin contained in the thrombus to cause blood clots. urokinase activity. It then converts plasminogen bound to fibrin molecules into plasmin, which appears to degrade fibrin.

In this way, when using this zymogen, fibrin (
Fibrinolysis can be expected to occur only on the solid phase (thrombus), and it holds great promise as a new type of fibrinolytic agent.

An example of the method for producing a plasminogen activator precursor according to the present invention will be shown below.

〔Example〕

Cultured human kidney cells were cultured for 3 days in a serum-free culture medium supplemented with 0.1% human serum albumin, the culture medium was centrifuged, and the supernatant was frozen and stored. The pooled culture supernatant was adjusted to pH 5.
5, and then brought into contact with CM-Sephadex C-50. After washing the column with 0.16M phosphate buffer (pH 5.5), 0.16M phosphate buffer (pH 8.
5) was used to elute the zymogen adsorbed on the column.

On the other hand, mouse BALB immunized with this zymogen in advance
Among the hybridomas made by fusing spleen cells of /c and mouse myeloma cells with polyethylene glycol,
Clones with high antibody production against this zymogen were selected. The anti-zymogen monoclonal antibody was recovered from the culture medium of this fused cell. This monoclonal antibody was
Br active compound Sepharose 4B (Pharm
acia).

0.1M phosphate buffer containing 0.4M NaCl (pH 7.
This monoclonal antibody column was equilibrated using 0) and contacted with the eluate containing the present zymogen. 0.1M phosphate buffer containing 0.4M NaCl (p
After washing the column with H7.0), the zymogen adsorbed on the column was washed with 0.2M glycine-H containing 0.5M NaCl.
Elution was performed with a Cl aqueous solution (pH 2.5). After the eluate is sterilized and filtered, it is lyophilized and the specific activity is at least 80.
000 U/mg of highly purified zymogen was obtained.

In addition, this purified product showed one band with a molecular weight of 50,000 by SDS-polyacrylamide gel electrophoresis.

〔Effect of the invention〕

By the method for producing a plasminogen activator precursor according to the present invention, plasminogen, which has high fibrinolytic activity,
Zymogens that can be converted into activators can be produced (separated, purified).

In addition, especially when the zymogen-producing cells are human kidney cells, the method of the present invention can increase the specific activity to at least 80,000.
0 IU/mg of zymogen can be produced.

[Brief explanation of the drawing]

Figure 1 shows the cDNA encoding human urokinase precursor.
The predicted amino acid sequence from A and the region identified from amino acid sequence analysis of this zymogen are shown. In the figure, solid lines indicate regions identified by amino acid sequence analysis, while dashed lines indicate unidentified regions. Cm-P,
C1-8 and L1-12 represent the reductively alkylated zymogen, its CNBr-degraded peptide, and its lysyl endopeptidase-degraded peptide, respectively. CPaseA (←) indicates a region identified from the C-terminal side using carboxyl peptidase A. Patent applicant: Midori Juji Co., Ltd. Patent attorney: Hajime Takashima

Claims (2)

[Claims] 1. A method for producing a plasminogen activator precursor, which comprises sequentially carrying out at least the following steps (a) and (d). (a) Cell culture supernatant producing this precursor at pH 4.5~
The present precursor is adsorbed by contacting with a cation exchanger under the conditions of 6.5. (b) The present precursor is eluted from the cation exchanger under conditions of pH 7.5 to 9.5. (c) The eluted fraction is brought into contact with the present precursor antibody column under conditions of pH 6 to 8 to adsorb the present precursor. (d) The precursor is eluted from the antibody column under conditions of pH 2 to 4. Claim 2: A protein that can be recovered from the culture medium of human kidney cells, has a molecular weight of about 50,000 Daltons as measured by SDS-polyacrylamide gel electrophoresis, does not undergo lower molecular weight when treated with a reducing agent, and Although it does not exhibit enzymatic activity itself, it expresses enzymatic activity by treatment with plasmin, and the specific activity at that time is at least 80,000 IU/mg.
Claims characterized in that a plasminogen activator precursor, which is a type of zymogen, is obtained (
1) Manufacturing method described.